A Single Dose of Beetroot Juice Does Not Change Blood Pressure Response Mediated by Acute Aerobic Exercise in Hypertensive Postmenopausal Women

e: To verify if acute intake of beetroot juice potentiates post-exercise hypotension (PEH) in hypertensive postmenopausal women. Methods: Thirteen hypertensive postmenopausal women (58.1 4.62 years and 27.4 4.25 kg/m 2 ) were recruited to participate in three experimental sessions, taking three di erent beverages: Beetroot juice (BJ), placebo nitrate-depleted BJ (PLA), and orange avored non-caloric drink (OFD). The participants performed moderate aerobic exercise training on a treadmill, at 65–70% of heart rate reserve (HRR), for 40 min. After an overnight fast, the protocol started at 07h when the rst resting blood pressure (BP) was measured. The beverage was ingested at 07h30 and BP was monitored until the exercise training started, at 09h30. After the end of the exercise session, BP was measured every 15 min over a 90-min period. Saliva samples were collected at rest, immediately before and after exercise, and 90 min after exercise

07h when the rst resting blood pressure (BP) was measured. The beverage was ingested at 07h30 and BP was monitored until the exercise training started, at 09h30. After the end of the exercise session, BP was measured every 15 min over a 90-min period. Saliva samples were collected at rest, immediately before and after exercise, and 90 min after exercise for nitrite (NO2 ) analysis. Results: There was an increase in salivary NO2 with BJ intake when compared to OFD and PLA. A slight increase in salivary NO2 was observed with PLA when compared to OFD (p<0.05), however, PLA resulted in lower salivary NO2 when compared to BJ (p<0.001). There were no changes in salivary NO2 with the OFD. Systolic and diastolic BP decreased (p<0.001) on all post exercise time points after all interventions, with no di erence between the three beverages. Conclusion: Acute BJ intake does not change PEH responses in hypertensive postmenopausal women, even though there is an increase in salivary NO2 . Keywords:nitrite; nitric oxide; hypertension; menopause; Post Exercise Hypotension Nutrients2019,11, 1327; doi:10.3390 /nu11061327 /journal/nutrients

Nutrients2019,11, 1327 2 of 13 1. Introduction There are several physiological changes in women's body during climacteric period, including reduction of estrogen levels and menstruation cessation (menopause). As estrogen is a cardioprotective hormone [1], subsequent to its reduction, there is an increased risk of developing cardiometabolic diseases, due to reduced vasodilatation, increased blood pressure (BP), oxidative stress, and in ammation [2]. Hypertension is one of the most prevalent cardiometabolic diseases after menopause [3], and pharmacological therapy is the most common treatment approach. However, changes in lifestyle, such as developing healthy dietary habits and performing exercise regularly, are important factors for hypertension prevention and control [3]. The practice of regular physical exercise can reduce resting BP chronically, and induce post-exercise hypotension (PEH), which is a reduction in BP below resting values after exercise training. This phenomenon is clinically relevant in the prevention and treatment of cardiovascular diseases, and in the reduction of cardiovascular events [4]. This cardiovascular regulatory response has been related to endothelium-derived nitric oxide (NO) [4,5]. Exercise-induced shear stress promotes the release of calcium by endothelial cells, from which the calcium binds to calmodulin and stimulates endothelial nitric oxide synthase (eNOS) to reduce L-arginine to NO, where NO is directly involved in vascular tone regulation and homeostasis [5]. During exercise, NO release induces vasodilation, increasing blood ow to skeletal muscles and regulating blood pressure [5]. In addition, consumption of foods containing inorganic nitrate (NO3 ) can increase NO bioavailability. This increase occurs by the initial conversion of NO3 into nitrite (NO2 ) in the mouth and stomach, through non-enzymatic reactions. The NO2 is then released into the systemic circulation for subsequent NO production [6], leading to improvements in hemodynamic regulation. Therefore, consumption of foods rich in NO3 may be a good strategy to improve BP regulation in target populations with an increased incidence of cardiometabolic diseases, especially when associated with exercise [7–12], since these two pathways of NO production can complement each other [6]. A common food that has a high NO3 concentration is beetroot, and some e ects observed with beetroot juice (BJ) consumption are related to improvement

a good strategy to improve BP regulation in target populations with an increased incidence of cardiometabolic diseases, especially when associated with exercise [7–12], since these two pathways of NO production can complement each other [6]. A common food that has a high NO3 concentration is beetroot, and some e ects observed with beetroot juice (BJ) consumption are related to improvement in blood ow, vasodilation, and reduction of BP [13–15]. The majority of studies that investigated BJ ingestion are related to sports and exercise performance [16–19], and only a few studies explored BJ consumption in parameters of health, in non-athletic populations, such as evaluating hemodynamic parameters in patients with chronic cardiorespiratory diseases [16,20]. These results are still inconclusive, since one did not nd PEH with BJ consumption [20], and the other found PEH only in diastolic BP (DBP) [16]. Moreover, there is a lack of studies evaluating BJ bene ts for hypertensive post-menopausal women. This population is of interest because there is a reduction in NO production, both due to hypertension and decreased estrogen levels. Therefore, the purpose of this study was to verify if BJ ingestion has additional e ects on PEH after one aerobic exercise session, and its relationship with salivary NO2 in hypertensive postmenopausal women. Our hypothesis was that BJ would improve blood pressure response after exercise in hypertensive postmenopausal women, due to an increase in the NO pathway, measured by salivary NO2 levels. 2. Materials and Methods 2.1. Participants The study intervention was conducted between June and September 2018, at the Laboratory of Cardiorespiratory and Metabolic Physiology of the Federal University of Uberl¥ndia, Uberl¥ndia, MG, Brazil. This study design was approved by the local Ethics Committee (70104717.0.0000.5152) and registered at form prior to admission.

Nutrients2019,11, 1327 3 of 13 For the inclusion criteria, participants were required to be: in post menopause (amenorrhea for at least 12 months and [FSH]>40 mIU/mL); diagnosed with hypertension, according to the 7th Brazilian Arterial Hypertension Directive [3], which is de ned by baseline blood pressure values greater than or equal to 140 mmHg for systolic BP (SBP) and 90 mmHg for DBP; aged between 50 and 70 years, and able to perform exercises on a treadmill. The exclusion criteria consisted of: use of hormonal therapy; history of food allergies that could compromise the study; sensitivity to NO3 ; history of stroke or acute myocardial infarction; diagnosis of Diabetes Mellitus; and smoking habits. Although all the volunteers used antihypertensive drugs, they were excluded if they were taking drugs of the -blocker class. All participants went through a cardiological evaluation with a specialist before the intervention, obtaining a certi cate to attest individual suitability for exercise practice. Exclusion criteria also applied to volunteers who failed to perform the protocol test, with some of the reasons, including intolerance to the exercise program, inability to ingest the juice, or inability to go through the fasting time. The number of volunteers required for this trial was calculated considering BP variation caused by BJ relative to placebo (PLA) as the main variable, with a variation of 5 4 mmHg de ned as an acceptable e ect [13]. Using online software (OpenEpi), considering a bilateral 95% con dence interval, and power analysis of 80%, a minimum of 11 people were needed for this study. After the calculation, 15 volunteers were recruited to participate in this study. Two out of the 15 volunteers were excluded from the study for inability to adapt to the study protocol, due to an abrupt decrease in blood pressure followed by initial syncope as of 15 min after the beginning of the rst exercise session. One of the volunteers had consumed BJ and the other PLA. The study volunteers answered both an anamnesis and a physical activity questionnaire (IPAQ short version). Anthropometric measures included: body mass (Filizola electronic scale); height ( xed

to an abrupt decrease in blood pressure followed by initial syncope as of 15 min after the beginning of the rst exercise session. One of the volunteers had consumed BJ and the other PLA. The study volunteers answered both an anamnesis and a physical activity questionnaire (IPAQ short version). Anthropometric measures included: body mass (Filizola electronic scale); height ( xed stadiometer Sanny); abdominal circumference (Filizola inelastic tape); and body composition (bioimpedance Inbody 230, Seoul, Coreia do Sul), assessed as previously described [21]. For the body composition measurement, all participants were instructed not to perform vigorous physical exercise 24 h before the test and to avoid alcohol and ca eine consumption 72 h before the test. 2.2. Study Design The present study was a double blind randomized, placebo-controlled, and crossover trial. The intervention comprised a total of three visits with a minimum wash-out interval of ve days in between visits. In each visit, one of the following beverages was taken: Placebo (PLA), non-caloric orange avor drink (OFD), or Beetroot Juice (BJ). Volunteers arrived in the laboratory at 07:00. and left at 11:40. FigureNutrients 2019, 11, x FOR PEER REVIEW 3 of 13 hormonal therapy; history of food allergies that could compromise the study; sensitivity to NO3 -; history of stroke or acute myocardial infarction; diagnosis of Diabetes Mellitus; and smoking habits. Although all the volunteers used antihypertensive drugs, they were excluded if they were taking drugs of the β-blocker class. All participants went through a cardiological evaluation with a specialist before the intervention, obtaining a certificate to attest individual suitability for exercise practice. Exclusion criteria also applied to volunteers who failed to perform the protocol test, with some of the reasons, including intolerance to the exercise program, inability to ingest the juice, or inability to go through the fasting time. The number of volunteers required for this trial was calculated considering BP variation caused by BJ relative to placebo (PLA) as the main variable, with a variation of 5 ± 4 mmHg defined as an acceptable effect [13]. Using online software (OpenEpi), considering a bilateral 95% confidence interval, and power analysis

juice, or inability to go through the fasting time. The number of volunteers required for this trial was calculated considering BP variation caused by BJ relative to placebo (PLA) as the main variable, with a variation of 5 ± 4 mmHg defined as an acceptable effect [13]. Using online software (OpenEpi), considering a bilateral 95% confidence interval, and power analysis of 80%, a minimum of 11 people were needed for this study. After the calculation, 15 volunteers were recruited to participate in this study. Two out of the 15 volunteers were excluded from the study for inability to adapt to the study protocol, due to an abrupt decrease in blood pressure followed by initial syncope as of 15 minutes after the beginning of the first exercise session. One of the volunteers had consumed BJ and the other PLA. The study volunteers answered both an anamnesis and a physical activity questionnaire (IPAQ short version). Anthropometric measures included: body mass (Filizola electronic scale); height (fixed stadiometer Sanny); abdominal circumference (Filizola inelastic tape); and body composition (bioimpedance Inbody 230, Seoul, Coreia do Sul), assessed as previously described [21]. For the body composition measurement, all participants were instructed not to perform vigorous physical exercise 24h before the test and to avoid alcohol and caffeine consumption 72h before the test. 2.2. Study Design The present study was a double blind randomized, placebo-controlled, and crossover trial. The intervention comprised a total of three visits with a minimum wash-out interval of five days in between visits. In each visit, one of the following beverages was taken: Placebo (PLA), non-caloric orange flavor drink (OFD), or Beetroot Juice (BJ). Volunteers arrived in the laboratory at 07:00. and left at 11:40. Figure 1 illustrates the experimental design of the sessions. Figure 1. Experimental design of the sessions. (C) Arrival at the laboratory; (*) Blood pressure measurement; (#) Saliva sample collection; (&) Beverage intake. During all intervention sessions, the volunteers arrived at 07:00, after eight hours of an overnight fast. BP was measured after 20 minutes of rest in a sitting position (07:20), every 30 minutes after the beverage intake until

Figure 1. Experimental design of the sessions. (C) Arrival at the laboratory; (*) Blood pressure measurement; (#) Saliva sample collection; (&) Beverage intake. During all intervention sessions, the volunteers arrived at 07:00, after eight hours of an overnight fast. BP was measured after 20 minutes of rest in a sitting position (07:20), every 30 minutes after the beverage intake until the beginning of the exercise (08:00, 08:30, and 09:00), and every 15 minutes for 90 minutes after the exercise session. Unstimulated saliva samples were collected after the 20 minutes of rest (07:20), immediately before (09:30) and after (10:10) exercise, and 90 minutes after the exercise session was finished (11:40). Heart rate (HR) was measured during the 20 minutes of rest (between 07:00 and 07:20), during the exercise, and during the 90 minutes after Figure 1. Experimental design of the sessions. (C) Arrival at the laboratory; (*) Blood pressure measurement; (#) Saliva sample collection; (&) Beverage intake. During all intervention sessions, the volunteers arrived at 07:00, after eight hours of an overnight fast. BP was measured after 20 min of rest in a sitting position (07:20), every 30 min after the beverage intake until the beginning of the exercise (08:00, 08:30, and 09:00), and every 15 min for 90 min after the exercise session. Unstimulated saliva samples were collected after the 20 min of rest (07:20), immediately before (09:30) and after (10:10) exercise, and 90 min after the exercise session was nished

Nutrients2019,11, 1327 4 of 13 (11:40). Heart rate (HR) was measured during the 20 min of rest (between 07:00 and 07:20), during the exercise, and during the 90 min after the exercise session (Polar ® RS800CX). The beverage intake took place 10 min after the rest period (07:30), allowing 15 min for the consumption of all the beverage content. The exercise session was 40 min long (from 09:30 to 10:10). During the sessions, the volunteers were allowed to drink water, but no other drink or food ingestion was permitted. They were instructed to avoid foods and drinks rich in NO3 24 h before the sessions and received a list with the following consumption restrictions: Green vegetables (amaranth, lettuce, cabbage, spinach, broccoli, celery, cauli ower, Chinese radish), beetroot or its juice, sausage, salami, ham, turkey breast, co ee, energy drinks, soft drinks, alcoholic beverages, and to avoid the use of mouthwashes. Before starting each intervention session, volunteers were questioned about these items. 2.3. Physical Exercise The exercise consisted of 40 min of continuous moderate intensity aerobic exercise on a treadmill, allowing the rst ve minutes to warm-up and the last two minutes to cool down. The treadmill speed could reach 5.5 km/h and the intensity increase was imposed by inclining the treadmill until the volunteer reached the zone between 65% and 70% of HR reserve (HRR) [22]. For the HRR calculation, we used the formula: maximum HR—resting HR. For the participants resting HR, we considered the minimum HR measured during the initial 20 min of rest on the rst intervention day, and the maximum HR was estimated by the formula: 220 age. HR was monitored during the exercise and the Borg Scale [23] was used to assess the subjective perception of exertion (RPE) for both dyspnea and lower limb fatigue. The measurements for HR and RPE were assessed every two minutes. Whenever the HR was found to be outside of the stipulated zone, the exercise load was readjusted. 2.4. Intake of Beetroot Juice and Placebo The intervention included three di erent juices: BJ, PLA and OFD, with one beverage assigned to

exertion (RPE) for both dyspnea and lower limb fatigue. The measurements for HR and RPE were assessed every two minutes. Whenever the HR was found to be outside of the stipulated zone, the exercise load was readjusted. 2.4. Intake of Beetroot Juice and Placebo The intervention included three di erent juices: BJ, PLA and OFD, with one beverage assigned to each intervention session. The order of beverage intake was randomly assigned for each of the volunteers using a randomized block design through the website (https: //www.random.org/lists/). For the randomization, codes were assigned to each volunteer, each beverage, and each session. Thereafter, the beverage each volunteer would drink on the rst session, second and third sessions were randomly allocated in this order. A researcher who did not participate in the data collection process was responsible for assigning a beverage to each code and blinding the drinks, adding each drink to its respective bottle labeled with the volunteer's name. The BJ had 20.78 mmol/kg of NO3 and was prepared using 35 mL of NO3 concentrated beetroot juice containing 400 mg of NO3 (Beet-It Sport Shot, James White Drinks Ltd., Ipswich, UK), which was diluted in 315 mL of distilled water with 6 g of non-caloric orange juice avored powder (Clight, Mondelez International, Inc., S¢o Paulo, Brazil), totalizing 350 mL of juice. The PLA had 3.86 mmol/kg of NO3 and was prepared by ltering the BJ on an ion exchange resin, capable of depleting the NO3 (PA101 OH-, Permution ® ) [24], similarly to a previous report [25]. Lastly, the OFD was a non-caloric orange avored drink, prepared using three grams of orange juice powder diluted in 350 mL of distilled water. BJ and PLA were identical in taste, while OFD was slightly di erent. Each volunteer received the designated drink in a sealed bottle with a lid and a dark straw, making it impossible to visualize or smell the bottle content. The study participants had 15 min to drink the entire beverage. 2.5. Measurements of Blood Pressure BP was measured in a sitting position, without visual or sound stimulation, using OMRON ®

di erent. Each volunteer received the designated drink in a sealed bottle with a lid and a dark straw, making it impossible to visualize or smell the bottle content. The study participants had 15 min to drink the entire beverage. 2.5. Measurements of Blood Pressure BP was measured in a sitting position, without visual or sound stimulation, using OMRON ® BP HEM-7113 automatic monitors. Prior to the BP measurement, 20 min of rest was required. BP was measured three times, always in the left arm, and the mean of these measures was calculated for data analysis.

Nutrients2019,11, 1327 5 of 13 2.6. Salivary Samples Collection and Analysis Saliva samples were collected using the spit method [26]. All samples were centrifuged at 3000 rpm for 15 min, the supernatant was separated and stored at 80 C until analysis. The NO2 concentration was used to estimate the bioavailability of NO by the Griess method [27]. 2.7. Statistical Analysis Statistical analyses were performed while researchers were blinded regarding the ingested beverage. Results are presented as mean standard deviation. The Shapiro-Wilk test was applied to verify data normality, and two-way ANOVA was used to analyze di erences between the time points (pre and post exercise) and the treatments (PLA, OFD, BJ), using the Bonferroni post hoc when necessary. The area under the curve (AUC) by the trapezoidal method was used to compare the temporal changes of BP and salivary NO2 , separately. One-way ANOVA was used for AUC and exercise analysis, and a Pearson correlation was used to assess BP and NO2 e ects. Statistical signi cance was set atp<0.05. All analyses were performed using SPSS version 20 (IBM SPSS, Chicago, IL, USA) and GraphPad Prism 6 (GraphPad Prism Inc., San Diego, CA, USA). 3. Results This study was completed by 13 postmenopausal women who were overweight, medically treated for hypertension, and physically active. The general characteristics of the volunteers are described in Table. There was no di erence in exercise intensity between the sessions. For PLA, OFD, and BJ sessions, the HR mean was 125.6 7.2; 126.1 7.8; 126.7 7.4 bpm (p=0.658); the treadmill inclination was 3.4 3.0; 3.5 2.7; 3.3 2.7% (p=0.715); the RPE of dyspnea was 3.7 0.85; 3.4 1.0; 3.4 1.0 (p=0.328); and the RPE of lower limb fatigue was 4.1 1.2; 3.9 1.12; 3.8 1.1 (p=0.642), respectively. Table 1.General characteristics of the participants. BMI: Body mass index. The general characteristics values are shown as mean standard deviation (SD), physical activity level and drugs are shown as: number of subjects (n) and percentage of the total number of subjects (%). General Characteristics (Mean SD) Age (years) 58.1 4.6 Body mass (kg) 69.9 9.2 Height (m) 1.57

The study investigates the effects of beetroot juice on blood pressure after exercise.